Both human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC) have the ability to differentiate into somatic cells. Thus, hESC and hiPSC differentiation offers an opportunity for therapy development, drug screening, disease modeling, and tissue replacement. However, developing well-defined conditions to generate pure populations of specific cell types is critical to achieve these goals. There are several neural induction methods that induce cell cultures to form neural stem cells (NSC) using spontaneous differentiation, chemical induction or mouse stromal feeder cells. NSC may be manually isolated and propagated as monolayer cultures for many passages. In principle, these cells may differentiate to neurons and glia, providing an endless supply of cells for in vitro and in vivo assays. Unfortunately, the robustness of these methods is hampered by batch-to-batch variability of isolated NSC. Moreover, differentiation of NSC often results in variable and heterogeneous cultures of neurons, glia and undifferentiated cells, which may impede downstream applications that require purified or defined cell populations, such as in vitro assays, transplantation and micro arrays. One possible solution to this problem is to identify cell surface markers expressed on NSC, glia and neurons to define and purify distinct cell types.
Cell surface marker expression has been described for the identification and isolation of many neural cell types by fluorescence-activated cell sorting FACS from embryonic and adult tissue from multiple species. The glycoprotein CD133 is a known stem/progenitor cell marker in many tissues and has been used to isolate NSC from human brain. The carbohydrate moiety CD15, also known as stage-specific embryonic antigen-1 or LeX, has been used to isolate NSC and radial glia from the sub-ventricular zone (SVZ) in mice. CD184, a G protein coupled receptor, was successfully used in combination with CD15 to isolate NSC from mouse embryonic forebrain and adult SVZ. CD24 is a cell adhesion molecule that has been used to isolate NSC from mouse brain by FACS. In addition, neural stem cells and neural progenitors have been isolated from human brain tissue using genetic promoter-reporters of neural stem cell markers.
Likewise, advancements have been made in the identification and isolation of hESC-derived neural cells by FACS. Pruszak et al., (Stem Cells 25: 2257-2268, 2007) reported that cultures of hESC differentiating to neural lineages may be assayed at different developmental stages with cell surface markers and that neurons could be enriched using an antibody to CD56 (NCAM). CD 184+/CD326− markers have been used to purify neural progenitors capable of differentiation into neurons from differentiating hESC. In addition, Peh et al. (Stem Cells Dev 18: 269-282, 2009) have reported the enrichment for neurosphere-forming NSC from neural induction cultures of hESC based on expression for CD133, CD15 and GCTM-2. Using a similar strategy, Golebiewska et al., (Stem Cells 27: 1298-1308, 2009) used CD133+/CD45−/CD340− to isolate NSC from differentiating hESC. Pruszak et al., (2009) demonstrated the utility of CD24, CD15 and CD29 as a surface maker code for isolating distinct cell populations, including NSC and a mixed population of neuroblasts and neurons from neural induction cultures of hESC.
Yuan et al., (PLoS One. 2011 Mar. 2; 6(3):e17540), incorporated herein by reference, describes methods based on cell surface signatures to isolate neural stem cells (NSC), neural differentiation cultures of pluripotent stem cells by FACS. The methods were used to isolate a population of NSC that was CD 184+/CD271−/CD44−/CD24+ from neural induction cultures of hESC and human induced pluripotent stem cells (hiPSC). Cell surface signature-based methods were also used to enrich NSC from multiple common neural induction culture systems. Yuan et al., further identified cell surface signatures for the isolation of neurons and glia from isolated cultures of isolated NSC cultures. A population of neurons that was CD184−/CD44−/CD15LOW/CD24+ and a population of glia that was CD 184+/CD44+ were subsequently purified from isolated cultures of differentiating NSC. The BD Stemflow™ Neural Cell Isolation Kit (BD Biosciences, San Jose, Calif.) is a reagent kit designed to allow the isolation of neural stem cells (NSCs) derived from human pluripotent stem cells or the isolation of neurons and glial cells from differentiated NSCs. This method reduces the amount of variability between NSC isolations as opposed to traditional methods like manual isolation but require the induction and isolation of NSC before isolation and enrichment of neurons in a culture. Methods are needed that provide for the identification and isolation of neurons derived from heterogeneous cell population and reduce the steps involved in neuron identification, isolation and enrichment.